PROJECT SUMMARY The growing global epidemic of metabolic disease is a pressing public health issue. As the prevalence of disorders such as obesity, insulin resistance, and nonalcoholic fatty liver continue to climb, the need for a thorough understanding of cellular lipid storage mechanisms has become increasingly imperative. Most metabolic disorders involve the aberrant accumulation of lipid in tissues such as the liver and heart, leading to devastating systemic health effects. Within cells, lipids are stored in cytosolic organelles called lipid droplets (LDs), which consist of a neutral lipid core of triacylglycerols and cholesterol esters bounded by a phospholipid monolayer. Associated with the monolayer are multiple regulatory proteins and enzymes that control the dynamic sequestration and release of the lipid reserves, allowing LD proteins to influence the metabolism of the entire cell. Although LD proteins have essential roles in maintaining cellular lipid homeostasis, little is known regarding the regulation of LD proteins themselves ? in particular, the pathways that control LD protein abundance. Although several studies report a role for the ubiquitin-proteasome system (UPS) in modulating LD protein levels, the identities of the required ubiquitination machinery (e.g. E3 ubiquitin ligases and E2 ubiquitin conjugating enzymes) and the pathways by which they exert control remain unclear. To address these fundamental questions, I propose 1) a genome-wide, fluorescence-based CRISPR/Cas9 screen to identify the degradation pathway of the LD protein perilipin-2 (PLIN2), and 2) follow-up studies to interrogate how impaired PLIN2 degradation impacts global cellular metabolism. I have characterized a human hepatoma Cas9-expressing fluorescent reporter cell line in which endogenous PLIN2 is tagged with GFP. Flow cytometry, Western blotting, and fluorescence microscopy analyses confirmed that PLIN2-GFP is expressed at endogenous levels, localizes to LDs, and is degraded by the proteasome. The validated PLIN2-GFP cell line was employed in a pilot screen of a 10-guide-per-gene lentiviral sublibrary of single guide RNAs (sgRNAs) enriched in UPS genes. This screen identified several candidate UPS factors that I hypothesize are involved in PLIN2 degradation. My proposed studies include the completion of a comprehensive, genome-wide screen, validation of candidate genes, characterization of PLIN2 degradation pathways, and examination of the functional consequences of impaired PLIN2 clearance. These studies will elucidate the mechanisms of LD protein regulation by the UPS, providing novel insights into how cells maintain lipid homeostasis. Knowledge of LD protein degradation pathways will allow for an understanding of how alterations in LD protein regulation contribute to the pathogenesis of metabolic disease.